LAB 7 – Fermentation & Cellular Respiration

LAB 7 ? Fermentation & Cellular Respiration

Objectives

1. Measure fermentation products produced by yeast. 2. Assess citric acid cycle activity in mitochondria.

INTRODUCTION

The cells of all living organisms require energy to keep themselves alive and fulfilling their roles. Where does this energy come from? The answer is energy released from molecules of the nucleotide adenosine triphosphate or ATP.

As you can see in the diagram to the left, the hydrolysis of ATP to ADP (adenosine diphosphate) and inorganic phosphate (Pi) is exergonic and thus releases energy which cells can use to do any number of things. Once hydrolyzed, ATP can be regenerated from ADP and Pi, though this is endergonic and thus requires energy. The energy needed to regenerate ATP is obtained from "food", whatever that may be.

The food we eat is first digested by enzymes as you learned in the previous lab. Once the polymers in your food (e.g., polysaccharides, triglycerides, protein) have been broken down by enzymes into monomers (e.g., monosaccharides such as glucose, fatty acids, amino acids), they enter the blood circulation and are delivered to the cells of the body. Within cells, the processes of fermentation and cellular respiration will further catabolize (break down) these molecules, harvesting the energy they contain for the synthesis of ATP.

Let us now take a brief look at fermentation and cellular respiration to see how each process produces ATP using energy released from molecules of glucose. Keep in mind that, although we are focusing on glucose, other molecules such as fatty acids can be used for the same purpose, though in slightly different ways.

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Part 1: FERMENTATION

To produce ATP from glucose, whether by fermentation or cellular respiration, cells must first partially break it down by glycolysis ("sugar" "separation"). The enzymes involved in glycolysis are located in the cell cytoplasm and sequentially break down each 6-carbon molecule of glucose to two 3-carbon molecules of pyruvate. In the process, enough energy is extracted to produce 2 molecules of ATP.

glucose (6-carbons) 2 pyruvate (3-carbons)

energy

2 ADP + 2 Pi

2 ATP

In conjunction with glycolysis, cells will carry out fermentation if there is no oxygen (O2) available. When you overexert yourself for example, your muscles do not receive enough oxygen and temporarily ferment glucose. In another familiar example, yeast will ferment when placed in an enclosed environment with a source of carbohydrate such as grapes (for making wine) or hops and barley (for making beer).

Interestingly, fermentation does not produce any additional ATP. What it does do is regenerate an important molecule needed for a particular step in glycolysis. This molecule is the electron carrier NAD+, which if depleted will bring a halt to glycolysis and ATP production, resulting in cell death. Fermentation therefore contributes to ATP production indirectly by allowing glycolysis, and the production of 2 ATP per glucose, to continue unhindered.

2 NAD+

2 NADH

2H-

2H-

2 NAD+

glucose 2 pyruvate

2 fermentation products

2 ADP + 2 Pi

2 ATP

Glycolysis

Fermentation

As shown above, NAD+, an empty electron carrier, is converted to NADH, a full electron carrier (the electrons being "carried" are associated with the hydrogen atom) during glycolysis. Fermentation is simply one or more biochemical steps that transfer the H in NADH and an extra electron to a molecule of pyruvate. As a result, NADH is restored to NAD+, which is needed for glycolysis, and pyruvate is converted to a "fermentation product" which can be a variety of things depending on the organism.

Animals, including human beings, produce lactic acid when their cells ferment. In organisms from other kingdoms the fermentation products can be quite different. Some bacterial species produce acetic acid (vinegar) when they ferment, whereas others produce acetone (the main ingredient in nail polish) or other organic molecules. In the Kingdom Fungi, single-celled yeasts

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when fermenting will produce CO2 and ethanol instead. This process, known as alcohol fermentation, is the basis for beer and wine production. Regardless of the fermentation products, the purpose of fermentation is always the same ? to regenerate NAD+ so that glycolysis can continue to produce 2 ATP per glucose without interruption.

In the following exercise you will investigate alcohol fermentation in yeast under different conditions and measure the production of one fermentation product ? CO2.

Exercise 1 ? Observing and Measuring Alcohol Fermentation in Yeast

1. You will use the following table to mix the proper amounts of water, yeast solution and corn syrup (a source of sugar) in small beakers. Be sure to add the yeast last. This will allow the reactions to begin at approximately the same time. Before you begin, review the experiment, write your hypothesis on your worksheet, and identify the independent and dependent variables as well as the control.

Water Corn syrup

Yeast

TOTAL

Tube #1 8 ml 4 ml 0 ml

12 ml

Tube #2 6 ml 4 ml 2 ml

12 ml

Tube #3 4 ml 4 ml 4 ml

12 ml

2. Once complete, transfer each mixture to a labeled saccharometer (do not overfill), gently tilting until no air is trapped inside the top of the tube. At this point you will begin the timing of your experiment (record on your worksheet).

3. At 5 minute intervals, record the volume of gas collected in each saccharometer on your worksheet. (NOTE: the saccharometers are graduated in ml)

4. Record the data on your worksheet, graph the data (be sure your X-axis is time and draw a separate curve for each tube), and answer any associated questions.

Part 2: CELLULAR RESPIRATION

While 2 ATP per glucose molecule is clearly better than nothing, it is not nearly enough to meet the energy needs of complex multicellular organisms such as plants and animals. To get the maximum ATP yield from molecules of glucose requires cellular respiration, which and produce up to 36 ATP per glucose molecule. In aerobic organisms, cellular respiration requires O2 (which is why we breathe!), hence the term aerobic respiration.

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The overall process of cellular respiration can be summarized in the following equation:

glucose

oxygen

C6H12O6 + 6 O2

carbon dioxide

water

energy

6 CO2

+ 6 H2O

36 ADP + 36 Pi

36 ATP

In eukaryotic cells, cellular respiration begins with glycolysis in the cytoplasm and continues in the mitochondria as outlined below:

The Citric Acid Cycle ? This is a biochemical pathway involved in breaking pyruvate down to CO2. In the process, energy rich electrons in hydrogen atoms are transferred to NAD+ and FAD producing NADH and FADH2. In addition, 2 ATP per original glucose are also produced.

Oxidative Phosphorylation ? This is the process by which the remaining 32 ATP molecules are produced involving two distinct stages:

? Electron Transport - electrons gathered by NADH and FADH2 during glycolysis and the citric acid cycle are used to produce an H+ gradient within mitochondria in a process that requires O2

? Chemiosmosis ? the H+ gradient produced by electron transport provides energy for ATP synthase to make 32 ATP per original glucose

The importance of O2 for cellular respiration cannot be overemphasized. O2 is the final electron acceptor in the electron transport chain. Without O2 electron transport does not occur, bringing cellular respiration to a halt, and the only option for ATP production is fermentation. This means 2 ATP per glucose instead of 36. The cell diagram below summarizes fermentation and cellular respiration in relation to O2 and where each process occurs in eukaryotic cells, and the number of ATP molecules produced.

no O2 Glycolysis

2 ATP (per glucose)

FERMENTATION

O2 present

CELLULAR RESPIRATION

Citric Acid Cycle

2 ATP (per glucose)

Oxidative Phosphorylation

32 ATP

(per glucose)

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In the next exercise you will detect the oxidation of succinate, a metabolic intermediate in the Citric Acid Cycle, as evidence of cellular respiration. Succinate dehydrogenase (SDH) is an enzyme in the Citric Acid Cycle which catalyzes the removal of 2 hydrogens from succinate (i.e., the oxidation of succinate) which are transferred to the electron carrier FAD. This yields the products fumarate and FADH2 as shown below:

succinate

FAD + succinate dehydrogenase FADH2 + fumarate

FADH2 in turn will donate the electrons from these 2 hydrogens to coenzyme Q in the electron transport chain. The compound DCPIP (di-chlorophenol-indophenol) is not normally found in cells, however when added to mitochondria it will substitute for coenzyme Q and receive electrons from FADH2. Before receiving the electrons (in its oxidized state) DCPIP is a blue color, however after receiving the electrons (being reduced by FADH2) DCPIP is colorless. Because of this color change, DCPIP is a good indicator of respiration as illustrated below.

fumarate SDH succinate

DCPIP DCPIP

(colorless)

In the next exercise you will add DCPIP to a mitochondrial suspension made from lima beans (yes, plants carry out cellular respiration too!) and detect the citric acid cycle step illustrated above by the loss of blue color in DCPIP.

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